GSM7049628: E2/E3/E4 5XFAD mice, 10weeks, HTO for demuxing; Mus musculus; RNA-Seq
Briefly, mice were anesthetized with a ketamine/xylazine cocktail and perfused with 25 ml of Ca2+/Mg2+-free DPBS (Sigma). Cortex and hippocampus were removed and placed in FACS buffer (PBS containing 5% FBS and 10 mM HEPES), minced with scissors, and incubated with 80 U/mL of collagenase D (Roche, 11088858001) at 37°C for 30 min. Collagenase was inactivated by adding 10 mM EDTA for an additional 5-min incubation at 37°C. Digested material was passed through a 70μm cell strainer, centrifuged at 1500rpm for 10min, pellet was then resuspended in 7mL of  38% Percoll followed by a centrifugation at 2000rpm for 30 min. Nonspecific binding to FC receptors was blocked by incubation with a CD16- and CD32-specific antibody (BD-Pharmingen 553141) for 15 min. Lastly, cells were washed and stained with an anti-CD45 antibody (Invitrogen 56-0451-82) and 0.05 µg/mL DAPI and, then washed and resuspended in FACS buffer. Cells were sorted on an LSR-II flow cytometer (Becton Dickinson) for singlet DAPI- CD45+ cells. Libraries were prepared according to manufacturer's instructions (3'v2, 5'v2, multiome; 10X Genomics). For 10-week and 20-week samples, hashed demultiplexing was performed using Cell Ranger multi, with read R2 and pattern 5PNNNNNNNNNN(BC)NNNNNNNNN. Samples from APOE2-bearing mice were barcoded with GGTCGAGAGCATTCA; samples from APOE3-bearing mice were barcoded with CTTGCCGCATGTCAT; samples from APOE4-bearing mice were barcoded with AAAGCATTCTTCACG.